Synthesis of estetrol via estrone derived steroids

ABSTRACT

A process is provided for the making of estetrol starting from a 3-A-oxy-estra 1,3,5(10),15-tetraen-17-one, wherein A is a C 1 -C 5  alkyl group, preferably a methyl group, or a C 7 -C 12  benzylic group, preferably a benzyl group. This process is particularly suitable to industry.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the synthesis of estetrol[estra-1,3,5(10)-trien-3,15α,16α,17β-tetraol; CAS Nr. 15183-37-6; forconvenience, this compound is referred to in this patent application as“estetrol”] via estrone derived steroids, preferably to the synthesis ofestetrol which is obtained with high yield and high purity.

BACKGROUND OF THE INVENTION

Estrogenic substances are commonly used in methods of HormoneReplacement Therapy (HRT) and methods of female contraception. Theseestrogenic substances can be divided in natural estrogens and syntheticestrogens. Examples of natural estrogens that have found pharmaceuticalapplication include estradiol, estrone, estriol and conjugated equineestrogens. Examples of synthetic estrogens, which offer the advantage ofhigh oral bioavailability include ethinyl estradiol and mestranol.

Recently, estetrol has been found effective as an estrogenic substancefor use in HRT, disclosure of which is given in the Applicant'sco-pending application WO 02/094276. Estetrol is a biogenic estrogenthat is endogeneously produced by the fetal liver during humanpregnancy. Other important applications of estetrol are in the fields ofcontraception, therapy of auto-immune diseases, prevention and therapyof breast and colon tumors, enhancement of libido, skin care, and woundhealing as described in the Applicant's co-pending applications WO02/094276, WO 02/094279, WO 02/094278, WO 02/094275, EP 2077272.9, EP2077273.7, WO 03/041718, WO 03/018026, EP 2077812.2, and EP 2077322.2.

The synthesis of estetrol and derivatives thereof on a laboratory scalebasis is known in the art: Fishman J., Guzik H., J. Org. Chem. 33,3133-3135 (1968); Nambara T. et al., Steroids 27, 111-121 (1976); orSuzuki E. et al., Steroids 60, 277-284 (1995).

Fishman J., Guzik H., J. Org. Chem. 33, 3133-3135 (1968) discloses asuccessful synthesis of estetrol from an estrone derivative (compound(III); cf. for a synthesis of compound (III) Cantrall, E. W., Littell,R., Bernstein, S. J. Org. Chem 29, 214-217 (1964)). In a first step, thecarbonyl group at C₁₇ of compound (III) was reduced with LiAlH₄ toestra-1,3,5(10),15-tetraene-3,17-diol (compound VIa) that was isolatedas the diacetate (compound VIb). Compound VIb was subjected tocis-hydroxylation of the double bond of ring D by using OsO₄ whichresulted into the formation ofestra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compoundIb) that under heating with K₂CO₃ in methanol produces estetrol (Scheme1).

The overall yield of this three step process is, starting from estronederivative III, only about 7%. It is worth noting that the protectedderivative 17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate(compound IV) could be cis-hydroxylated to its 15α,16α-diol derivative(compound Va), but that thereafter the dioxolane group could not beremoved (p-toluene sulfonic acid in acetone at room temperature) or thatthe hydrolysis (aqueous sulfuric acid in warm dioxane) of the dioxolanegroup resulted in a mixture containing a multitude of products (Scheme2).

Nambara T. et al., Steroids 27, 111-121 (1976) discloses anothersynthesis of estetrol wherein estrone is the starting material. Thecarbonyl group of estrone is first protected by treatment with ethyleneglycol and pyridine hydrochloride followed by acetylation of the hydroxygroup at C₃. The next sequence of steps involved a bromination/basecatalyzed dehydrobromination resulting into the formation of17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol (compound IVa). Thiscompound IVa was subsequently acetylated which produced17,17-ethylenedioxyestra-1,3,5(10),15-tetraene-3-ol-3-acetate (compoundIVb). In a next step, the dioxolane group of compound IVb was hydrolysedby using p-toluene sulfonic acid to compound Vb, followed subsequentlyby reduction of the carbonyl group at C₁₇ (compound Vc) and oxidation ofthe double bond of ring D thereby formingestra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compoundVIb). See Scheme 3.

Suzuki E. et al., Steroids 60, 277-284 (1995) also discloses thesynthesis of estetrol by using compound Vb of Nambara T. et al. asstarting material. The carbonyl group at C₁₇ of this compound was firstreduced followed by acetylation yieldingestra-1,3,5(10),15-tetraene-3,17-diol-3,17-diacetate (compound 2b). Thelatter was subjected to oxidation with OsO4 which providedestra-1,3,5(10)-triene-3,15α,16α,17β-tetraol-3,17-diacetate (compound3b) in 46% yield.

According to the Nambara T. et al. and Suzuki E. et al., the synthesisof estetrol can be performed with a yield of approximately 8%, startingfrom estrone.

Poirier D., et al., Tetrahedron 47, 7751-7766 (1991) discloses thefollowing compounds which were prepared according to methods that havebeen used to prepare similar compounds:

Dionne, P. et al., Steriods 62, 674-681 (1997) discloses the compoundshown above wherein R is either methyl or t-butyldimethylsilyl.

Magnus, P. et al., J. Am. Chem. Soc. 120, 12486-12499 (1998) disclosesthat the main methods for the synthesis of α,β-unsaturated ketones fromsaturated ketones are (a) halogenation followed by dehydrohalogenation,(b) utilising sulphur or selenium derivatives, (c) DDQ and (d) utilizingpalladium(II) complexes.

Furthermore, it has also been found that by following the prior artmethods mentioned above, estetrol of high purity was obtained only inlow yield when using an acetyl group as a protecting group for the3-hydroxy group of estra-1,3,5(10),15-tetraen-3-ol-17-one, in particularbecause its sensitivity to hydrolysis and solvolysis. In particular, thelability of the acetyl group lead not only to an increased formation ofbyproducts during the reactions, but also during chromatography andcrystallisation for purification of intermediate products when proticsolvents such as methanol were used. Therefore, it is difficult toisolate purified estetrol and intermediates thereof in good yield.

Additionally, the reduction of the carbonyl group at C₁₇ with LiAlH₄proceeds with a low selectivity since various amounts of β-estradiol(estra-1,3,5(10)-trien-3,17β-diol) are obtained as well. Obviously, theformation of such a by-product reduces the yield as well as the purityof the desired product which requires additional purification steps.

The prior art methods also employ stoichiometric amounts of OsO₄ in theoxidation step that is known to be a toxic and expensive compound.Consequently, the use of such a reagent is undesired in view of safetyand operational costs.

Accordingly, it is an object of the present invention to provide asynthesis route for estetrol whereby high yields and high purities ofestetrol are obtained.

Still accordingly, there is a need for a synthesis of estetrol whereinthe production of by-products is limited. i.e. preferably less than itsdetection level.

It is a preferred object of the invention to provide a synthesis ofestetrol wherein good yield and good purity of estetrol are provided.

By a good yield, it is meant a yield of at least 10%, preferably higherthan 10%, more preferably of at least 12.5%, starting from estrone(100%).

By a good purity, it is meant a purity of at least 97%, preferably of atleast 98%, more preferably of at least 99%. Preferably, singleimpurities are not allowed to exceed 1%. Also preferred is thatβ-estradiol is not allowed to exceed the detection level.

For the purpose of the present invention, determination of purity ismade by HPLC-MS. The following conditions are used:

HPLC-MS is performed using a Hewlett Packard 1100 series:

-   -   Column: Discovery C18 (150×4.6 mm) Supelco    -   Mobile phase: Solution A:Solution B=70:30 (5 min)→(10 min)→10:90        (5 min)    -   Flow: 1 mL/min    -   UV: 280 nm    -   Temp: 22° C.    -   MS: API-ES negative    -   Solution A: 9.65 g NH₄OAc, 2250 mL H₂O, 150 mL MeOH, 100 mL        CH₃CN    -   Solution B: 9.65 g NH₄OAc, 250 mL H₂O, 1350 mL MeOH, 900 mL        CH₃CN

It has now been found that protecting the 3-OH group ofestra-1,3,5(10),15-tetraen-3-ol-17-one by an C₁-C₅ alkyl group,preferably a methyl group, or a C₇-C₁₂ benzylic group, preferably abenzyl group, fulfils such a need. Indeed, it has been found that theuse of a more stable protective group such as a C₁-C₅ alkyl group,preferably a methyl group, or a C₇-C₁₂ benzylic group, preferably abenzyl group, on the 3-OH group is not cleaved at an undesired stage ofthe synthesis. Therefore the formation of by-products is limited and thepurification of intermediates is more practical.

In this patent application the term “alkyl” includes linear, branchedand cyclic alkyl groups such as methyl, ethyl, n-propyl, i-propyl,c-propyl, n-butyl, s-butyl, t-butyl, c-butyl, n-pentyl, s-pentyl,t-pentyl, c-pentyl and methylcyclobutyl. Additionally, the C₇-C₁₂benzylic group has to be understood as a benzyl group that may besubstituted with one or more substituents at the ortho, meta and/or paraposition of the aromatic nucleus, wherein the substituents are aliphaticgroups, optionally substituted by one or more heteroatoms and/or halogenatoms that do not adversely interfere with the synthetic process. As isobvious to a skilled person in the art, the alkyl and benzylic groupsare intended as a protecting group and these groups must therefore berelatively easy to add and relatively easy to remove under suchconditions that do not have an adverse effect on the molecular structureof the estrone derived steroid molecules.

Because of the selected protecting groups which are used and the yieldand purity obtained, it appeared that the synthesis disclosed in thispatent application can be suitably transposed to an industrial scale.This represents a particular advantage in comparison to the currentlab-scale syntheses which have been disclosed in the prior art and whichhamper from several disadvantages as disclosed above. Indeed, a problemwith industrial syntheses are the quantities of chemicals as well as thetoxicity and hazardous properties thereof which are involved, thusmaking the prior art lab-scale methods not transposable to an industrialscale. The reason behind such impossible replication is that usually theknown method either does not provide a sufficient yield, i.e. at least10% to be considered economically feasible from an industrial point ofview and/or produce by-product(s) which necessitates at least asubsequent purification step, thus raising the cost of the method.

Accordingly, it is also another preferred object of the invention toprovide a method which is suitable for use in industry.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the present invention, a process isprovided for the obtainment of1,3,5(10)-estratrien-3,15α,16α,17β-tetraol which comprises the steps of:

-   1) 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one wherein A is a    protecting group;-   2) reduction of the 17-keto group;-   3) protection of the reduced carbonyl function of the    3-A-oxy-estra-1,3,5(10),15-tetraen-17-one;-   4) oxidizing the alkene bond of the cyclopentenol moiety of the    acetylated 3-A-oxy-estra-1,3,5(10),15-tetraen-17-ol; and-   5) removing the protecting groups;    wherein the protecting group A is selected from an C₁-C₅ alkyl    group, preferably a methyl group, or a C₇-C₁₂ benzylic group,    preferably a benzyl group.

Hence, according to this first aspect of the invention, a process isprovided for the preparation ofestra-1,3,5(10)-trien-3,15α,16α,17β-tetraol (1) which comprises thesteps of:

-   1) converting estrone (7) into    3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is a    protecting group;-   2) reduction of the 17-keto group of    3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) to    3-A-oxy-estra-1,3,5(10),15-tetraen-17β-ol (5);-   3) protection of the 17-OH group of    3-A-oxy-estra-1,3,5(10),15-tetraen-17β-ol (5) to    3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4), wherein C is a    protecting group;-   4) oxidizing the carbon-carbon double bond of ring D of    3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to protected    estetrol (3); and-   5) removing the protecting groups, wherein preferably protecting    group A is removed first to form 17-OC protected estetrol (2) and    subsequently protecting group C is removed to form estetrol (1);    wherein the protecting group A is selected from an C₁-C₅ alkyl    group, preferably a methyl group, or a C₇-C₁₂ benzylic group,    preferably a benzyl group, and the protecting group C is selected    from monofunctional aliphatic hydroxyl protecting groups, said    monofunctional aliphatic hydroxyl protecting groups being preferably    selected from the group consisting of C₁-C₅ carboxylates wherein the    alkyl group of the carboxylates is as defined above, said protecting    group C being most preferably acetyl.

The process according to this first aspect of the invention is shown inScheme 4.

In another aspect of the invention, there is provided the use of theobtained compound as estrogenic substance, preferably for cosmeticand/or therapeutic applications selected from hormone replacementtherapy, contraception, therapy of autoimmune diseases, prevention andtherapy of breast and colon tumors, enhancement of libido, skin care,and wound healing.

DETAILED DESCRIPTION OF THE INVENTION

According to one aspect of the invention, a process is provided for theobtainment of 1,3,5(10)-estratrien-3,15α,16α,17β-tetraol. The inventionprocess comprises the steps of:

-   1) providing a 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one, wherein A    is a protecting group selected from an C₁-C₅ alkyl group, preferably    a methyl group, or a C₇-C₁₂ benzylic group, preferably a benzyl    group.

This may be achieved by methods known in the art for making suchcompounds such as given in J. Am. Chem. Soc. 79, 2005-2009 (1957),“14-Isoestrone Methyl ether and its identity with totally syntheticmaterial” by W. S. Johnson and W. F. Johns (A is methyl); Biosci.Biotech. Biochem. 60, 411-414 (1996), “Synthesis of(14β,17α)-14-Hydroxy- and (14β,17α)-14-Dihydroxyestradiols and TheirActivities” by M. Sakakibara and A. O. Uchida.

Still, another process of obtainment has been found effective forproviding the 3-A-oxy-estra-1,3,5(10),15-tetraene-17-one (6) wherein Ais a protecting group selected from a C₁-C₅ alkyl group, preferably amethyl group, or a C₇-C₁₂ benzylic group, preferably a benzyl group.This process comprises the steps of:

-   -   Step i)—protecting the phenol function of estrone by alkylation        to obtain a protected estrone;    -   Step ii)—protecting the carbonyl function of the protected        estrone obtained in step a),    -   Step iii)—forming an alkene bond in the C₁₅-C₁₆ position of the        protected estrone obtained in step b); and    -   Step iv)—deprotecting the carbonyl function.

This process for the preparation of3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) comprises a thirdembodiment of the invention and comprises the following steps:

-   (a1) conversion of the 3-OH group of estrone (7) into a 3-AO group    to form 3-A-oxy-estra-1,3,5(10)-trien-17-one (8);-   (b1) conversion of the 17-keto group of    3-A-oxy-estra-1,3,5(10)-trien-17-one (8) into a protected keto group    to form 3-A-oxy-17-D-estra-1,3,5(10)-triene (9);-   (c1) halogenation of C₁₆ of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9)    to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is a    halogen atom selected from the group chloride, bromide and iodide    and wherein X is preferably bromide;-   (d1) dehalogenation of 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10)    to 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and-   (e1) deprotection of the protected keto group of    3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form    3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6),    wherein A is selected from an C₁-C₅ alkyl group, preferably a methyl    group, or a C₇-C₁₂ benzylic group, preferably a benzyl group, and    wherein D is ethylene dioxy.

The process according to this third embodiment of the invention isdepicted in Scheme 5.

Step (a)

Estrone (7) is a product which is commercially available from Acros,Aldrich under the tradename estrone. Other suppliers of estrone areAndard-Mount Company Ltd., Diosynth B. V., Productos Quimicos NaturalesS. A. de C. V.-Proquina, Schering A G, Mistsubishi Chemical Corporation.

The protection of the 3-OH group by alkylation is typically carried outby reacting estrone with a component selected from an alkylating agent,preferably a C₁-C₅ alkyl halogenide, preferably a methyl halogenide, ora C₇-C₁₂ benzylic halogenide, preferably benzyl halogenide. Preferably,the halogen atom of the alkylating agent is bromide, chloride or iodide,most preferably bromide or iodide. According to the present invention,the most preferred alkylating agent is benzyl bromide or methyl iodide,wherein benzyl bromide is even more preferred than methyl iodide.According to the invention, however, it is possible to use a dialkylsulphate instead of a C₁-C₅ alkyl halogenide, wherein the alkyl groupscontain 1-5 carbon atoms and wherein the alkyl groups are preferablymethyl (i.e. that the preferred dialkyl sulphate is dimethyl sulphate).Nevertheless, according to this embodiment of the invention, the mostpreferred alkylating agent is benzyl bromide.

According to this third embodiment of the present invention, it is inparticular preferred to first suspend estrone (7) and potassiumcarbonate in a mixture of dichloromethane (DCM)/methanol. A 1:1 mixtureof DCM/methanol is preferred. The alkylating agent C₇-C₁₂ benzylichalogenide, preferably benzyl bromide, is added and the resultingmixture is refluxed for a period of 8-16 hours. It is preferred toreflux the mixture for 16 hours. The reaction mixture is then cooled toRoom Temperature (RT). The product is isolated by filtering off thesolids. The filter cake is washed with a protic solvent, preferablymethanol. The filtrate is concentrated to give a suspension which isfiltered and washed with heptanes to give the product as a white solid.The product can be purified by recrystallisation from a mixture of DCMand MeOH to obtain a white crystalline solid, wherein the preferredratio of DCM:MeOH is 1:2.

In particular, it is preferred to first suspend estrone (7) andpotassium carbonate in DMF. The C₁-C₅ alkyl halogenide, preferablymethyl iodide, is added with cooling, keeping the temperature between18° and 22° C. The resulting mixture is stirred for a period of time atRT, preferably for 5 days. The reaction mixture is poured into water andstirred for 2 hours. The product is collected by filtration and washedwith water. The product is dried to give a white crystalline solid.

Step b)

The protection of the 17-keto group is preferably carried out byreacting 8 with ethylene glycol using an acid catalyst such as p-toluenesulfonic acid, HCl pyridine, sulfuric acid or acetic acid and a solventselected from dimethoxyethane, toluene, benzene, trimethyl orthoformateor triethyl orthoformate. More preferably the reaction is performed withethylene glycol, triethyl orthoformate and p-toluenesulfonic acid.

In particular, it is preferred to suspend 8 in a mixture of triethylorthoformate and ethylene glycol in a preferred volume ratio of 4:3,more preferably 2:1. Subsequently, p-toluenesulfonic acid is added andthe reaction mixture is stirred for a period of time at 35° C.Preferably, after 1-16 hours, preferably about 3 hours, the mixture ispoured into a mixture of ice/water and pyridine. After stirring for 1 hthe product is collected by filtration. It is washed with water anddried to yield the product as a white solid.

Alternatively, it has also been found that steps (a) and (b) canadvantageously be performed simultaneously or sequentially without theneed for purification and/or isolation of the intermediate productswhilst still providing an end-product with good yield and purity. Thisis particularly advantageous for use in industry where the reduction ofthe number of process step provides both an economical advantage and asimplification of the process by eliminating the need for an additionalstep like purification and/or isolation between the two steps.

If the process is made sequentially, the order for carrying thesynthesis is preferably by having first the protection of the 3-OH group(step (a)) and then protection of the 17-keto group (step (b)). Still,it is preferred to first have step (b) and then step (a) carried out.Indeed, by use of this order, the formation of by-products has beenfound reduced upon industrial process.

Hence, according to a fourth embodiment of the present invention, thereis provided a process for the obtainment of a3-A-oxy-estra-1,3,5(10),15-tetraen-17-one wherein A is the protectinggroup selected from an C₁-C₅ alkyl group, preferably a methyl group, ora C₇-C₁₂ benzylic group, preferably a benzyl group, which processcomprises the steps of:

-   -   Step ia) protecting the carbonyl function of estrone to obtain a        protected estrone;    -   Step iia) protecting the phenol function of the protected        estrone obtained in step ia) by alkylation,    -   Step iii) forming an alkene bond in the C₁₅-C₁₆ position of the        protected estrone obtained in step iia);    -   Step iv)—deprotecting the carbonyl function;        wherein steps ia) and iia1) are performed simultaneously or        sequentially without purification and/or isolation of the        obtained intermediate product.

This process for the preparation of3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) comprises a fourthembodiment of the invention and comprises the following steps:

-   (a2) conversion of the 17-keto group of estrone (7) into a protected    keto group to form 17-D-estra-1,3,5(10)-trien-3-ol (12);-   (b2) conversion of the 3-OH group of    17-D-estra-1,3,5(10)-trien-3-ol (12) into a 3-AO group to form    3-A-oxy-17-D-estra-1,3,5(10)-trien-17-one (9);-   (c2) halogenation of C₁₆ of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9)    to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is a    halogen atom selected from the group chloride, bromide and iodide    and wherein X is preferably bromide;-   (d2) dehalogenation of 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10)    to 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and-   (e2) deprotection of the protected keto group of    3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form    3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6),    wherein A is selected from an C₁-C₅ alkyl group, preferably a methyl    group, or a C₇-C₁₂ benzylic group, preferably a benzyl group, and    wherein D is ethylene dioxy.

Preferably, this is achieved by stirring a mixture of estrone (7),ethylene glycol and triethyl orthoformate to which is then added acatalytic amount of acid, preferably p-toluene sulfonic acid. Thereaction temperature is then raised to between about 40° and about 60°C., preferably to about 45° C. The slurry is stirred at that sametemperature until completion of the reaction, i.e. protection of thecarbonyl function of estrone. The conversion is checked with HPLC. Tothe slurry, a solution of base, preferably sodium methoxide in methanolis added resulting in a clear yellow solution. By use of such a base,the 3-OH group is completely deprotonated which advantageously allowsthe use of the less reactive but more economical C₇-C₁₂ benzylicchloride, preferably benzyl chloride, in the alkylation process. Thetemperature is raised to 65° C. This high temperature further enables agood crystallisation of the product. Although lower temperatures such asdown to 20° C. can be used, it is believed that the low temperaturewould incur a lower reactivity, thus longer reaction times and probablyincomplete conversions. C₇-C₁₂ benzylic chloride, preferably benzylchloride, is then added over a few minutes, such as 5 minutes, uponwhich the solution becomes turbid and slowly thickens into a slurry.After 1.5 hours the conversion is checked with HPLC, usually aconversion of >95% is observed, which is sufficient for furtherprocessing.

The mixture is allowed to cool to 20° C. while stirring, and then thesolid product is isolated by filtration. The solid is then washed anddried.

According to the present invention, the fourth embodiment is morepreferred than the third embodiment.

Step 3)

The formation of the carbon-carbon double bond in ring D is preferablycarried out by steps (c2) and (d2) defined above.

The halogenation is carried out with a halogenating agent. Preferredhalogenating agents are selected from bromine, phenyltrimethylammoniumperbromide or pyridinium bromide perbromide. A more preferredhalogenating agent for use herein is pyridinium bromide perbromide. Thesolvent is selected from CHCl₃, dioxane, dimethoxyethane, ethyleneglycol or THF. The preferred solvent is THF without any co-solvent.

In particular, it is preferred to dissolve the previously obtainedcompound 9 in dimethoxyethane, which is subsequently added to a solutionof the brominating reagent in a mixture of ethylene glycol anddimethoxyethane. The resulting mixture is stirred until completion ofthe reaction. Preferably after 16 hours the product is isolated. Asolution of sodium thiosulfate pentahydrate in water is added to thereaction mixture. The product is extracted with an organic solvent,preferably dichloromethane. The extract is dried using sodium sulphateand the solvents are evaporated to obtain a sticky oil which canadvantageously be used without further purification.

However, it is even more preferred to dissolve compound 9 in pure THFand to perform the reaction at room temperature for less than two hours,followed by removal of the THF by distillation and adding a solvent thatis essential not miscible with water, preferably toluene. Water can thenbe removed from the product 10 by azeotropic distillation. Before thenext step is performed, the toluene solution of 10 is concentrated todryness and the solvent to be used in the next step is added.

The dehydrohalogenation reaction is carried out by using a base selectedfrom potassium tert-butoxide, DBU (1,8-diazabicylo[5.4.0]undec-7-ene) orpotassium hydroxide and is preferably potassium tert-butoxide. Thesolvent is selected from benzene, xylene, methanol or DMSO. The morepreferred base and solvent for use in this step are respectivelypotassium tert-butoxide and dimethyl sulfoxide (DMSO).

In particular, it is preferred to add a suspension of the previouslyobtained 10 compound in DMSO to a solution of potassium tert-butoxide inDMSO. The resulting mixture is then stirred until completion of thereaction. Preferably after about 2 hours the reaction mixture is pouredinto a mixture of ice and water. The product is extracted with anorganic solvent, preferably DCM. The extract is dried using sodiumsulfate and the solvents are evaporated to obtain a sticky oil which canbe used without further purification.

However, it is even more preferred to perform the dehydrohalogenationstep for less than one hour and to perform the extraction with tolueneat about 60° C. Furthermore, the toluene solution of compound 11 ispreferably dried by azeotropic distillation before the next step iscarried out.

Step 4)

Deprotection of the carbonyl function is preferably carried out by acomponent selected from p-toluenesulfonic acid, pyridiniump-toluenesulfonate, and pyridinium chloride, preferablyp-toluenesulfonic acid monohydrate. More preferably, the deprotection isperformed using p-toluenesulfonic acid monohydrate in the presence ofaqueous acetone as solvent.

In particular, it is preferred to add p-toluenesulfonic acid monohydrateto a solution of the previously obtained compound in aqueous acetone,preferably with 10-20% water. The mixture is stirred until completion ofthe reaction. Preferably after about 3 hours, DCM and saturated aqueoussodium bicarbonate are added. After separating the layers, the aqueouslayer is extracted with DCM. The combined extracts are washed with brineand concentrated to give a suspension. The product is collected byfiltration and is washed with organic solvents, preferably with coldacetone and heptane. The product can be purified by recrystallization.

However, it is even more preferred to perform this deprotection step inaqueous acetone (water content about 10%) during which the product 6crystallises from the solution. To enhance the crystallisation process,water is added after completion of the reaction which provides nicecrystals that are easily collected by filtration and which eliminatesthe necessity of further purification, e.g. by recrystallisation.

The obtained 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) isadvantageously used in the process for the obtainment of estetrol.

2) Reduction of the 17-keto Group

Reduction of the 17-keto group is preferably performed by reacting3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) with a reducing agentselected from the group of metal hydride compounds, said group of metalhydride compounds preferably comprising LiAlH₄, AlH₃, NaBH₄, NaBH(OAc)₃,ZnBH₄, and NaBH₄/CeCl₃. Most preferably the metal hydride compound isNaBH₄/CeCl₃. More preferred reducing agents for use herein are thosethat will provide a chemo- and stereo-selective reduction of the 17-ketogroup in favour of the P position. For that reason, the most preferredchemo- and stereo-selective reducing agent for use herein is NaBH₄ incombination with CeCl₃ hydrate, preferably the heptahydrate.

In particular, it is preferred to suspend3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) and CeCl₃ heptahydrate ina mixture of a protic solvent, preferably MeOH and THF and to stir themixture for about 1 h at room temperature. A preferred volume ratio ofMeOH to THF is 2:1 to 4:1. Then the mixture is cooled, preferably to0°-5° C., and NaBH₄ is added in small portions maintaining thetemperature below 8° C. After a period of time, preferably 2 hours, 1 NNaOH and DCM are added. After 30 minutes of stirring, the layers areseparated and the aqueous layer is extracted with DCM. The combinedorganic extracts are dried with sodium sulphate and concentrated to givethe product as a white solid.

However, it is even more preferred to quench the reaction mixture withan acid, preferably 2 N HCl, to remove the solvents by distillationunder vacuum at about 30° to about 40° C. and to add toluene.Preferably, the temperature is then raised to about 70° C. to inducephase separation. The organic phase is then separated, washed with anaqueous solution of Na₂CO₃ and water. The final organic phase is driedby azeotropic distillation, cooled to about 50° C. and used for the nextstep.

3) Protecting the reduced carbonyl function of the3-A-oxy-estra-1,3,5(10),15-tetraen-17-one, i.e. protection of the 17-OHgroup of 3-A-oxy-estra-1,3,5(10),15-tetraen-17β-ol (5) to form3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4), wherein C is aprotecting group.

The 17-OH group is protected by preferably selected by acetylation usinga reagent selected from acetic anhydride or acetyl chloride. Preferably,acetic anhydride is used.

In particular, it is preferred to treat a solution of the compound inpyridine with acetic anhydride and 4-dimethylaminopyridine. The mixtureis stirred for a period of time. Preferably after 2 hours at roomtemperature the volatiles are removed. The residue is dissolved in ethylacetate (EtOAc) and the resulting solution is washed with water andbrine. The solution is dried using sodium sulphate and concentrated togive the crude product. Recrystallization from a mixture of organicsolvents, preferably ethyl acetate, heptane and ethanol gives theproduct as a white solid.

However, since 4-dimethylaminopyridine is toxic and difficult to removeby distillation, it is more preferred to perform the reaction with atrialkylamine, preferably triethylamine, and an acetyl halide (about twoequivalents), preferably acetyl chloride (about 1.5 equivalent) intoluene at about 25° to about 60° C., preferably about 40° to about 50°C. The work up is then performed by washing with water, aqueous acid andaqueous base. Purification of the product is then achieved bycrystallisation, i.e. by removing the toluene by distillation,dissolving the crude product in ethyl acetate and heating this solutionto about 70° to about 80°. To this heated solution, small portions ofethanol are added to induce crystallisation (preferred ratio of ethylacetate to ethanol is about 1 to about 8).

4) Oxidizing the alkene bond of the cyclopentenol group of theacetylated 3-A-oxy-estra-1,3,5(10),15-tetraen-17-ol, i.e. oxidizing thecarbon-carbon double bond of ring D of3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to protected estetrol(3)

The oxidation of carbon-carbon double bond inn ring D is carried with anoxidising agent providing selective cis-hydroxylation of thecarbon-carbon double bond. Preferably, the oxidising agent is osmiumtetroxide and more preferably the oxidising agent is osmium tetroxideimmobilized on PVP (OsO₄—PVP) that is used in a catalytic amount (cf. G.Cainelli, M. Contento, F. Manesclachi, L. Plessi, Synthesis, 45-47(1989)) in combination with a co-oxidant selected fromtrimethylamine-N-oxide, N-methyl morpholine-N-oxide or hydrogenperoxide, preferably trimethylamine-N-oxide. More preferably, OsO₄—PVPand trimethylamine-N-oxide are used with TBF as the solvent.

In particular, it is preferred to add OsO₄—PVP to a heated solution ofthe compound prepared in the previous step in THF. Preferably, theaddition is performed at 50° C. followed by the addition oftrimethylamine-N-oxide. Preferably, the addition oftrimethylamine-N-oxide is performed portion wise during 1 hour. Themixture is stirred at this temperature for a period of time. Preferably,after 12 hours the mixture is cooled to room temperature and filtered.The volatiles are removed and the residue is dissolved in ethyl acetateand water is added. The aqueous layer is acidified and the layers areseparated. The aqueous layer is extracted with ethyl acetate. Thecombined extracts are dried with sodium sulphate and concentrated. Theresulting residue is triturated with heptanes and ethyl acetate to givethe product as a white precipitate that is filtered off. The product ispurified by recrystallization from a mixture of organic solvents,preferably ethyl acetate, heptane and ethanol to give the product as awhite solid.

5) Removing the Protecting Groups A and C

Removal of the protecting groups is also an important aspect of thepresent invention process. Indeed, it has been found that not allprotective groups can be removed without adverse effects on the obtainedproduct. Hence, where for example a methyl group is used as theprotective group for the 3-OH group, removal with pyridine.HCl has beenfound to lead to decomposition of the product.

Accordingly, it has been found that removal of the protecting C₁-C₅alkyl group is preferably performed using BBr₃ without leading to majordecomposition of the product.

Removal of the protective C₇-C₁₂ benzylic group is preferably beperformed using catalytic hydrogenation conditions (Pd./H₂) as is wellknown to the person skilled in the art.

In particular, it is preferred to dissolve the protected estrone(protected estetrol (3)) in a protic solvent, preferably methanol. Acatalytic amount of 10% Pd on carbon is added as a preformed suspensionin methanol and the mixture is placed under an atmosphere of hydrogen,preferably 1 atmosphere. After stirring the mixture for 3 hours at roomtemperature it is filtered over Celite. The filtrate is concentrated togive 17-OC protected estetrol (2) as a white solid.

Removal of protecting group C is effective using a protic solvent suchas methanol and a base, preferably K₂CO₃, to yield estetrol.

In particular, it is preferred to dissolve the compound obtained in theprevious step in methanol. Potassium carbonate is added and the mixtureis stirred for 2 hours at room temperature. Then the volatiles areevaporated and water and chloroform are added. The mixture isneutralized with 0.1 N HCl and the product is collected by filtration.It is then washed with water and dried to give estetrol as a whitesolid.

Alternatively, the order of the two deprotection steps above can bereversed. Thus, the complete deprotection can be accomplished by firstdeprotection of protecting group C followed by catalytic hydrogenationto remove protecting group A where A is a protective C₇-C₁₂ benzylicgroup. The procedures are identical to the ones described above.However, the first order of the deprotection steps that is describedhereinbefore is preferred over the latter, i.e. that according to theinvention it is preferred to first remove protecting group A andsubsequently protective group C.

According to a most preferred embodiment of this step, the deprotectionreactions, i.e. the removal of A and C, are performed in a single stepif A is a protective C₇-C₁₂ benzylic group. Preferably, compound 3 isdissolved in a C₁-C₃ alkyl alcohol, preferably methanol, and subjectedto hydrogenation at room temperature over night. Thereafter, thesolution of compound 2 is preferably used in the subsequent step, i.e.the removal of C as described above. Work up of the reaction mixture isthen preferably carried out by concentrating the solution to about 20%of its original volume and by adding an amount of water approximatelyequal to the volume of the concentrated solution. Thereafter,concentrated acid, preferably concentrated HCl, is added dropwiseresulting in a white suspension which is filtered off, washed with waterand dried.

Compounds and intermediate products that are in particular preferredaccording to the present invention are:

-   -   the compound according to formula 2, wherein C is acetyl.    -   compounds according to formula 3, wherein A is methyl or benzyl,        preferably benzyl, and wherein C is acetyl.    -   compounds according to formula 4, wherein A is methyl or benzyl,        preferably benzyl, and wherein C is acetyl.    -   compounds according to formula 5, wherein A is methyl or benzyl,        preferably benzyl.    -   compounds according to formula 8, wherein A is methyl or benzyl.    -   compounds according to formula 9, wherein A is methyl or benzyl,        preferably benzyl, and D is ethylene dioxy.    -   compounds according to formula 10, wherein A is methyl or        benzyl, preferably benzyl, D is ethylene dioxy and X is bromo.    -   compounds according to formula 11, wherein A is methyl or        benzyl, preferably benzyl, and D is ethylene dioxy.        Applications

In another aspect of the present invention is provided the use of theproduct as obtainable by the invention process for the manufacture of apharmaceutical composition, preferably for use in a method selected froma method of hormone replacement therapy, a method of treating vaginaldryness, a method of contraception, a method of enhancing libido, amethod of treating skin, a method of promoting wound healing, and amethod of treating or preventing a disorder selected from the groupconsisting of autoimmune diseases, breast tumours and colorectaltumours.

In another aspect of the present invention is provided thecosmetic/aesthetic use of the product as obtainable by the inventionprocess for treating skin.

EXAMPLES

The following are non-limited synthesis examples for the synthesis ofestetrol according to the invention:

The following methods and materials for determination were used:

1H NMR spectra were recorded on a Varian 200 MHz apparatus in CD₃OD.

HPLC-MS was performed using a Hewlett Packard 1100 series:

Column: Discovery C18 (150×4.6 mm) Supelco

Mobile phase: Solution A:Solution B=70:30 (5 min)→(10 min)→10:90 (5 min)

Flow: 1 mL/min

UV: 280 nm

Temp: 22° C.

MS: API-ES negative

Solution A: 9.65 g NH4OAc, 2250 mL H2O, 150 mL MeOH, 100 mL CH3CN

Solution B: 9.65 g NH4OAc, 250 mL H2O, 1350 mL MeOH, 900 mL CH3CN

DSC was measured using a Mettler Toledo DSC822 apparatus.

Example 1 3-Benzyloxy-estra-1,3,5(10)-trien-17-one (compound 6,A=benzyl)

To a suspension of estrone (7; 100 g, 0.370 mol) and K₂CO₃ (160 g, 1.16mol) in DCM/MeOH (800 mL, 1:1 v/v ratio) at room temperature was addedbenzyl bromide (132 mL, 1.10 mol) in one portion. The resulting mixturewas refluxed for 16 h (50% conversion after 4 h according to TLC). Thereaction mixture was cooled to RT and solids were filtered off. Thefilter-cake was washed with MeOH. The solution was concentrated (to atotal volume of ca. 300 mL). The precipitate that had formed wascollected by filtration and washed with heptanes to give a white solid.The filtrate was concentrated further (to a total volume of 100 mL) andtriturated with heptane. The resulting precipitate was filtered off andcombined with the first batch of product. The product (153 g, max 0.370mol) still contained traces off benzyl bromide but was used withoutfurther purification. The product can be purified by recrystallizationfrom DCM/MeOH (1/2). TLC: R_(f)=0.5 (heptanes/ethyl acetate=4/1);HPLC-MS: 91%; ¹H-NMR (200 MHz, CDCl₃) δ 7.60-7.24 (m, 5H), 7.49 (d, 1H,J=8.4 Hz), 6.87 (dd, 1H, J₁=2.6 Hz, J₂=8.4 Hz), 6.82 (d, 1H, J=2.4 Hz),5.12 (s, 2H), 3.05-2.90 (m, 2H), 2.66-2.01 (m, 5H), 1.77-1.47 (m, 8H),0.99 (s, 3H) ppm.

Example 2 17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10)-trien-17-one(compound 4; A=benzyl, C=ethylene dioxy)

3-Benzyl-estrone (compound 6, A=benzyl; 153 g (crude), max. 0.370 mol)was suspended in a mixture of triethyl orthoformate (320 mL) andethylene glycol (160 mL). p-TsOH monohydrate (5 g, 26.3 mmol) was addedand the resulting pinkish suspension was stirred for 3 h at 35° C. (TLCindicated complete conversion after 1.5 h). The mixture was cooled toRT, poured into a mixture of ice-water (2 L) and pyridine (40 mL). Theresulting precipitate was collected by filtration and washed with water(150 ml). The remaining white solid was dried azeotropically bystripping with toluene (2×200 mL) to afford the product (153 g, max.0.370 mmol) as white crystalline material. TLC: R_(f)=0.3(heptanes/ethyl acetate=9/1); ¹H-NMR (200 MHz, CDCl₃) δ 7.60-7.24 (m,5H), 7.29 (d, 1H, J=8.4 Hz), 6.86 (dd, 1H, J₁=2.6 Hz, J₂=8.4 Hz), 6.80(d, 1H, J=2.4 Hz), 5.11 (s, 2H), 4.03 (m, 4H), 3.05-2.90 (m, 2H),2.46-1.28 (m, 13H), 0.96 (s, 3H) ppm.

Example 3 17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10)-trien-17-one(compound 4; A=benzyl, C=ethylene dioxy)

A reaction flask equipped with mechanical stirrer, thermometer, nitrogenpurge, condenser and dropping funnel was used for the process. The flaskwas charged with 27 g (100 mmol) of estrone, 50 ml (55 g, 9 equivalents)of glycol and 24 g of triethylorthoformate. The resulting mixture wasstirred. 0.5 g of toluenesulfonic acid was added and the reactiontemperature was raised to 45° C. At about 35-40° C. an exothermic wasobserved. The slurry is stirred for 1 hour at 45° C. The conversion ischecked with LC. Usually after 1 hour almost complete conversion isobserved. To the slurry a solution of sodium methoxide in methanol (30%wt.; 1.1 equivalents) is added from the dropping funnel resulting in aclear yellow solution. The temperature is raised to 65° C. and 15 g ofbenzyl chloride is added over 5 minutes. Within a few minutes thesolution becomes turbid and slowly thickens into a slurry. After 1.5hours the conversion is checked with LC, usually a conversion of >95% isobserved, which is sufficient for further processing.

The mixture is allowed to cool to 20° C. while stirring, and then thesolid product is isolated by filtration. The solid is washed withmethanol (2*30 ml) and dried under atmospheric conditions.

An amount of 33-34 g of product is obtained with an organic purity of>97%.

Example 416-Bromo-17,17-ethylenedioxy-3-benzyloxy-estra-1,3,5(10)-triene-17-one(compound 10, X=Br, A=benzyl, B=ethylene dioxy)

Pyridinium bromide perbromide (120 g, 375 mmol, 1.44 equiv) wasdissolved in a mixture of ethylene glycol (120 mL) and ethylene glycoldimethyl ether (200 mL). 3-Benzyl-estrone ethylene glycol acetal(compound 4; A=benzyl, C=ethylenedioxy; 153 g (crude), max. 0.370 mol)was dissolved in ethylene glycol dimethyl ether (400 mL) andsubsequently added to the brominating reagent within 5 minutes. Themixture became yellow immediately and was stirred for 16 h at RT (TLCshowed the reaction to be converted to 50% after 2 h). A solution ofNa₂S₂O₃.5H₂O (205 g, 0.83 mol) in water (700 mL) was added to thereaction mixture. DCM (1 L) was added and the layers were separated. Theaqueous layer was extracted with DCM (2×200 mL). The combined organiclayers were washed with water (300 mL) and brine (300 mL), dried(Na₂SO₄) and concentrated in vacuo to yield the brominated product (180g, max. 0.370 mol) as a yellow solid which was used without furtherpurification for the next step. TLC: R_(f)=0.25 (heptanes/ethylacetate=9:1); HPLC-MS: 2 diasteromers (together 85%) minor byproductspresent; ¹H-NMR (200 MHz, CDCl₃) δ 7.60-7.20 (m, 5H), 7.27 (d, 1H, J=8.4Hz), 6.85 (dd, 1H, J₁=2.6 Hz, J₂=8.6 Hz), 6.80 (d, 1H, J=2.4 Hz), 5.10(s, 2H), 4.63 (m, 1H), 4.08 (m, 4H), 2.93 (m, 2H), 2.41-1.38 (m, 11H),0.98 (s, 3H) ppm.

Example 5 17,17-Ethylenedioxy-3-benzyloxy estra-1,3,5(10),15-tetraene(compound 11; A=benzyl, B=ethylene dioxy)

Potassium tert-butoxide (180 g, 1.6 mol) was dissolved in DMSO (600 mL)and a suspension of16-Bromo-17,17-ethylenedioxy-3-benzyloxy-estra-1,3,5(10)-triene-17-one(compound 10, X=Br, A=benzyl, B=ethylenedioxy; 180 g (crude), max. 0.370mol) in DMSO (600 mL) was added at RT within 5 min. The temperature roseto 45° C. during the addition. The colour of the reaction mixtureimmediately changed to dark brown. The reaction mixture was stirred for2 h during which the temperature fell to 25° C. It was poured intoice/water (2 L) and extracted with DCM (2×1 L, 2×300 mL). The organiclayers were combined, washed with water (300 mL) and brine (300 mL) anddried with Na₂SO₄. The solution was concentrated in vacuo to give thecrude product (147 g, max. 0.370 mmol) as a brown oil which was usedwithout further purification for the next step. TLC: R_(f)=0.35(heptanes/ethyl acetate=9/1); ¹H-NMR (200 MHz, CDCl₃) δ 7.60-7.44 (m,5H), 7.27 (d, 1H, J=8.4 Hz), 6.86 (dd, 1H, J₁=2.6 Hz, J₂=8.4 Hz), 6.80(d, 1H, J=2.4 Hz), 6.33 (dd, 1H, J₁=1.6 Hz, J₂=7.4 Hz), 5.82 (dd, 1H,J₁=3.4 Hz, J₂=6.0 Hz), 5.10 (s, 2H), 4.03 (m, 4H), 2.95 (m, 2H),2.56-1.40 (m, 9H), 1.04 (s, 3H) ppm.

Example 6 3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-one (compound 6;A=benzyl)

To a solution of 17,17-Ethylenedioxy-3-benzyloxyestra-1,3,5(10),15-tetraene (compound 11; A=benzyl, B=ethylenedioxy; 147g, max 0.370 mol) in acetone (0.9 L) and water (100 mL) at RT was addedp-TsOH monohydrate (4.8 g, 25 mmol). The mixture was stirred for 3 h atRT (According to TLC the reaction was complete after 1 h and aprecipitate had formed). DCM (1.2 L) and saturated aqueous NaHCO₃solution (300 mL) were added. The mixture was stirred vigorously. Thelayers were separated and the aqueous layer was extracted with DCM (300mL). The combined organic layers were washed with brine (300 mL) andconcentrated until precipitation started (volume of appr. 300 mL, T=50°C.). The precipitate was filtered off, washed with cold acetone andhexanes to afford the product as an off-white solid which was purifiedby recrystallization from acetone to give a white solid (58 g, 0.162mol, 44% over 5 steps). (purity according to HPLC-MS: 94%). Theremaining mother-liquor still contained 40% of product according toHPLC-MS. TLC: R_(f)=0.3 (heptanes/ethyl acetate=4:1); DSC: Mp. 161.9° C.(purity 91.7%); ¹H-NMR (200 MHz, CDCl₃) δ 7.70 (dd, 1H, J₁=1.6 Hz,J₂=6.0 Hz), 7.60-7.40 (m, 5H), 7.26 (d, 1H, J=8.8 Hz), 6.86 (dd, 1H,J₁=2.6 Hz, J₂=8.8 Hz), 6.84 (d, 1H, J=2.4 Hz), 6.17 (dd, 1H, J₁=3.8 Hz,J₂=6.6 Hz), 5.12 (s, 2H), 3.01 (m, 2H), 2.62-1.64 (m, 9H), 1.18 (s, 3H)ppm.

Example 7 3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-ol (compound 5;A=benzyl)

To a solution of 3-benzyl-dehydroestrone (compound 6; A=benzyl; 58 g,162 mmol) in a mixture of MeOH (900 mL) and THF (200 mL) at roomtemperature was added CeCl₃ heptahydrate (66.4 g, 178 mmol). Afterstirring for 1 h the mixture was cooled to 0-5° C. using an ice/waterbath. Then NaBH₄ (12.2 g, 324 mmol) was added in small portionsmaintaining a temperature below 8° C. After stirring for 2 h at 0-5° C.(TLC showed the reaction to be complete) 1 N NaOH (300 mL) and DCM (1 L)were added and the mixture was stirred for ½ h at room temperature. Thelayers were separated and the aqueous layer was extracted with DCM (200mL). The organic layers were combined, dried (Na₂SO₄) and concentratedin vacuo to give an off-white solid (55.0 g, 152.8 mmol, 94%) TLC:R_(f)=0.25 (heptanes/ethyl acetate=4:1); HPLC-MS: 93% β-isomer, 2%α-isomer; DSC: Mp. 149.7° C., purity 96.6%; ¹H-NMR (200 MHz, CDCl₃) δ7.48 (m, 5H), 7.27 (d, 1H, J=8.4 Hz), 6.85 (dd, 1H, J₁=2.8 Hz, J₂=8.6Hz), 6.81 (d, 1H, J=2.4 Hz), 6.10 (d, 1H, J=5.8 Hz), 5.79 (dd, 1H,J₁=1.8 Hz, J₂=3.4 Hz), 5.11 (s, 2H), 4.48 (d, 1H, J=7.6), 2.96 (m, 2H),2.46-1.64 (m, 9H), 0.93 (s, 3H) ppm.

Example 8 17-Acetyloxy-3-benzyloxy-estra-1,3,5(10),15-tetraene (compound4; A=benzyl, C=acetyl)

A solution of 3-Benzyloxy-estra-1,3,5(10),15-tetraen-17-ol (compound 5;A=benzyl; 55.0 g, max. 153 mmol) in pyridine (400 mL) was treated withAc₂O (50 mL, 0.53 mol) and 4-dimethylaminopyridine (1.5 g, 12.3 mmol).The mixture was stirred for 2 h at room temperature (TLC showed thereaction to be complete). It was concentrated in vacuo. The residue wasdissolved in EtOAc (400 mL), washed with water (200 mL) and brine (150mL), dried (Na₂SO₄) and concentrated in vacuo to yield a yellow solid(54.0 g, 49.8 mmol, 88%). The product was purified by recrystallizationfrom heptanes/EtOAc/EtOH (1:0.5:1) to afford a white solid (45.0 g, 112mmol, 73%) TLC: R_(f)=0.6 (heptanes/ethyl acetate=4/1); HPLC-MS: 98%β-isomer, 1% α-isomer, 1.3% β-estradiol; DSC: Mp. 122.8° C., purity99.8%; ¹H-NMR (200 MHz, CDCl₃) δ 7.44 (m, 5H), 7.27 (d, 1H, J=8.4 Hz),6.86 (dd, 1H, J₁=2.6 Hz, J₂=8.4 Hz), 6.80 (d, 1H, J=2.6 Hz), 6.17 (d,1H, J=5.8 Hz), 5.78 (dd, 1H, J₁=1.4 Hz, J₂=3.2 Hz), 5.45 (m, 1H), 5.11(s, 2H), 2.96 (m, 2H), 2.40-1.54 (m, 10H), 2.18 (s, 3H), 0.93 (s, 3H)ppm.

Example 9 17-Acetyl-3-Benzyl estetrol (compound 3; A=benzyl, C=acetyl)

OsO₄ on PVP (9 g, ˜5% w/w OsO₄ on PVP, prepared according to Cainelli etal. Synthesis, 45-47 (1989) was added to a solution of17-Acetyloxy-3-benzyloxy-estra-1,3,5(10),15-tetraene (compound 4;A=benzyl, C=acetyl; 45 g, 112 mmol) in THF (450 mL) and the mixture washeated to 50° C. Trimethylamine-N-oxide dihydrate (24.9 g, 224 mmol) wasadded portion-wise over 2 h. After stirring for 36 h at 50° C. (TLCshowed the reaction to be complete) the reaction mixture was cooled toroom temperature. The solids were filtered off, washed with THF (100 mL)and the filtrate was concentrated. The residue was taken up in EtOAc(250 mL) and water (250 mL) was added. The aqueous layer was acidifiedwith 1 N HCl (ca. 10 mL). The layers were separated and the aqueouslayer was extracted with EtOAc (150 mL). The organic layers werecombined, dried (Na₂SO₄) and concentrated in vacuo. The residue wastriturated with heptanes/EtOAc (1:1, 100 mL), stirred for 2 h and theresulting white precipitate was filtered off to give the product as awhite solid (41 g, 94 mmol, 84%). The product was purified byrecrystallization from heptanes/ethyl acetate/EtOH (2:1:1) three timesto afford a white solid (21 g, 48.2 mmol, 43%). HPLC-MS: 99.5%βαα-isomer; DSC: Mp. 159.3° C., purity 98.7%; ¹H-NMR (200 MHz, CDCl₃) δ7.49 (m, 5H), 7.27 (d, 1H, J=8.4 Hz), 6.84 (dd, 1H, J₁=2.6 Hz, J₂=8.4Hz), 6.81 (d, 1H, J=2.4 Hz), 5.11 (s, 2H), 4.45 (d, 1H, J=4.4), 4.11 (m,3H), 3.12 (m, 1H) 2.95 (m, 2H), 2.46-1.64 (m, 10H), 2.24 (s, 3H), 0.93(s, 3H) ppm.

Example 10 17-Acetyl estetrol (compound 2; C=acetyl)

To a solution of 17-acetyl-3-benzyl estetrol (compound 3; A=benzyl,C=acetyl; 21 g, 48.2 mmol) in MeOH (600 mL, HPLC-grade) was added apreformed suspension of 10% Palladium on activated carbon (2 g) inmethanol (50 mL). The mixture was placed under an atmosphere of H₂ at 1atm and stirred for 24 h (TLC showed the reaction to be completed) atroom temperature. It was filtered over Celite® and the filter cake waswashed with MeOH (200 mL). The filtrate was concentrated in vacuo togive 17-acetyl estetrol as a white solid (15 g, 43.4 mmol, 90%). TLC:R_(f)=0.2 (heptanes/ethyl acetate=1/1); HPLC-MS: 99.2%, DSC: Mp. 212.2°C., purity 98.9%; ¹H-NMR (200 MHz, CD₃OD) δ 7.14 (d, 1H, J=8.0 Hz), 6.60(dd, 1H, J₁=2.6 Hz, J₂=8.8 Hz), 6.56 (d, 1H, J=2.4 Hz), 4.81 (dd, 1H,J₁=3.4 Hz, J₂=6.4 Hz), 4.07 (m, 3H), 3.12 (m, 1H), 2.85 (m, 2H),2.37-1.37 (m, 10H), 2.18 (s, 3H), 0.91 (s, 3H) ppm.

Example 11 Estetrol

17-Acetyl-estetrol (compound 2; C=acetyl; 15 g, 43.4 mmol) and K₂CO₃ (6g, 43.4 mmol) were suspended in MeOH (500 mL, HPLC-grade) and stirredfor 4 h at room temperature (TLC showed the reaction to be complete).The solvents were evaporated in vacuo. Water (200 mL) and CHCl₃ (70 mL)were added and the mixture was stirred and neutralized with 0.1 N HCl(50 mL). The product was collected by filtration, washed with water (100mL) and CHCl₃ (100 nL) to give estetrol as a white solid (12.2 g, 40.1mmol, 92.5%, overall yield from estrone 10.8%) after drying at 40° C. inan air-ventilated oven. TLC: R_(f)=0.05 (heptanes/ethyl acetate=1/1);HPLC-MS: 99.1%, DSC: Mp. 243.7° C., purity 99.5%; ¹H-NMR (200 MHz,CD₃OD) δ 7.14 (d, 1H, J=8.6 Hz), 6.61 (dd, 1H, J₁=2.6 Hz, J₂=8.4 Hz),6.56 (d, 1H, J=2.4 Hz), 4.83 (m, 1H), 3.93 (m, 3H), 3.50 (d, 1H, J=5.2),3.38 (m, 2H), 2.84 (m, 2H), 2.32 (m, 3H), 1.97 (m, 1H), 1.68-1.24 (m,5H), 0.86 (s, 3H) ppm.

1-31. (canceled)
 32. A process for the preparation of estra-1,3,5(10)-trien-3,15α,16α,17β-tetraol (1), comprising the steps of: 1) converting estrone (7) into 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), wherein A is a protecting group; 2) reduction of the 17-keto group of 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6) to 3-A-oxy-estra-1,3,5(10),15-tetraen-17β-ol (5); 3) protection of the 17-OH group of 3-A-oxy-estra-1,3,5(10),15-tetraen-17β-ol (5) to 3-A-oxy-17-C-oxy-extra-1,3,5(10),15-tetraene (4), wherein C is a protecting group; 4) oxidizing the carbon-carbon double bond of ring D of 3-A-oxy-17-C-oxy-estra-1,3,5(10),15-tetraene (4) to protected estetrol (3); and 5) removing the protecting groups, wherein protecting group A is removed first to form 17-OC protected estetrol (2) and subsequently protecting group C is removed to form estetrol (1); wherein the protecting group A is selected from the group consisting of a C₁-C₅ alkyl group and a C₇-C₁₂ benzylic group and the protecting group C is selected from monofunctional aliphatic hydroxyl protecting groups.
 33. The process according to claim 32, wherein the protecting group is a C₇-C₁₂ benzylic group.
 34. The process according to claim 32, wherein the protecting group is a benzyl group.
 35. The process according to claim 32, wherein the protecting group C is selected from monofunctional aliphatic hydroxyl protecting groups.
 36. The process according to claim 35, wherein the monofunctional aliphatic hydroxyl protecting group is acetyl.
 37. The process according to claim 32, wherein the reduction of the carbonyl group is carried out using a reducing agent selected from the group of metal hydride compounds.
 38. The process according to claim 37, wherein the metal hydride compound is selected from the group consisting of LiAlH₄, NaBH₄, NaBH(OAc)₃, ZnBH₄, and NaBH₄/CeCl₃.
 39. The process according to claim 38, wherein the metal hydride compound is NaBH₄ in combination with CeCl₃ hydrate.
 40. The process according to claim 32, wherein the oxidation of the carbon-carbon double bond in ring D is carried out with an oxidizing agent comprising osmium tetroxide.
 41. The process according to claim 40, wherein the oxidizing agent is osmium tetroxide immobilized on PVP (OsO₄—PVP).
 42. The process according to claim 32, wherein the oxidization of the carbon-carbon double bond in ring D is carried out with a catalytic amount of OsO₄—PVP.
 43. The process according to claim 42, wherein the OsO₄—PVP is used in combination with a co-oxidant.
 44. The process according to claim 43, wherein the co-oxidant is selected from the group consisting of trimethylamine-N-oxide, N-methyl morpholine-N-oxide and hydrogen peroxide.
 45. The process according to claim 44, wherein the co-oxidant is trimethylamine-N-oxide.
 46. The process according to claim 32, wherein the protective C₇-C₁₂ benzylic group is removed by catalytic hydrogenation conditions.
 47. The process according to claim 46, wherein the catalytic hydrogenation conditions comprise a hydrogenation reaction using Pd on activated carbon under a hydrogen atmosphere.
 48. The process according to claim 32, wherein the protective C₁-C₅ alkyl group is removed by using BBr₃.
 49. A process for the preparation of 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6), comprising the steps of: (a1) converting the 3-OH group of estron (7) into a 3-AO group to form 3-A-oxy-estra-1,3,5(10)-trien-17-one (8); (b1) converting the 17-keto group of 3-A-oxy-estra-1,3,5(10)-trien-17-one (8) into a protected keto group to form 3-A-oxy-17-D-estra-1,3,5(10)-triene (9); (c1) halogenating C₁₆ of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9) to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10), wherein X is a halogen atom selected from the group consisting of chloride, bromide and iodide; (d1) dehalogenating 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and (e1) deprotecting the protected keto group of 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6); wherein A is selected from a C₁-C₅ alkyl group or a C₇-C₁₂ benzylic group and wherein D is ethylene dioxy.
 50. The process according to claim 49, wherein the halogen atom is bromide.
 51. The process according to claim 49, wherein A is a methyl group.
 52. The process according to claim 49, wherein A is a benzyl group.
 53. A process for the preparation of 3-A-oxy-estra-1,3,5(10),15-tetraene-17-one (6), comprising the steps of: (a2) converting the 17-keto group of estron (7) into a protected keto group to form 17-D-estra-1,3,5(10)-trien-3-ol (12); (b2) converting the 3-OH group of 17-D-estra-1,3,5(10)-trien-3-ol (12) into a 3-AO group to form 3-A-oxy-17-D-estra-1,3,5(10)-trien-17-one (9); (c2) halogenating C₁₆ of 3-A-oxy-17-D-estra-1,3,5(10)-triene (9) to form 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) wherein X is a halogen atom selected from the group consisting of chloride, bromide and iodide; (d2) dehalogenating 3-A-oxy-16-X-17-D-estra-1,3,5(10)-triene (10) to 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11); and (e2) deprotecting the protected keto group of 3-A-oxy-17-D-estra-1,3,5(10),15-tetraene (11) to form 3-A-oxy-estra-1,3,5(10),15-tetraen-17-one (6); wherein A is selected from a C₁-C₅ alkyl group or a C₇-C₁₂ benzylic group, and wherein D is ethylene dioxy.
 54. The process according to claim 53, wherein the halogen atom is bromide.
 55. The process according to claim 53, wherein A is a methyl group.
 56. The process according to claim 53, wherein A is a benzyl group.
 57. The process according to claim 49, wherein the protected keto group D is formed by converting the 17-keto group with ethylene glycol.
 58. The process according to claim 53, wherein the protected keto group D is formed by converting the 17-keto group with ethylene glycol.
 59. The process according to claim 49, wherein steps (e1) and (e2) are carried out in the presence of a component selected from the group consisting of p-toluenesulfonic acid, pyridinium p-toluenesulfonate and pyridinium chloride.
 60. The process according to claim 49, wherein steps (e1) and (e2) are carried out in the presence of p-toluenesulfonic acid.
 61. The process according to claim 49, wherein steps (e1) and (e2) are carried out in the presence of p-toluenesulfonic acid monohydrate using aqueous acetone as solvent.
 62. The process according to claim 53, wherein steps (e1) and (e2) are carried out in the presence of a component selected from the group consisting of p-toluenesulfonic acid, pyridinium p-toluenesulfonate and pyridinium chloride.
 63. The process according to claim 53, wherein steps (e1) and (e2) are carried out in the present of p-toluenesulfonic acid.
 64. The process according to claim 53, wherein steps (e1) and (e2) are carried out in the presence of p-toluenesulfonic acid monohydrate using aqueous acetone as solvent.
 65. A pharmaceutical composition, comprising a carrier and the product obtainable by the method of claim
 32. 66. A method of hormone replacement therapy, of treating vaginal dryness, of contraception, of enhancing libido, of treating skin, of promoting wound healing or of treating or preventing an autoimmune disease, breast tumours or colorectal tumours, comprising administering the pharmaceutical composition of claim 65 to a subject in need thereof.
 67. A cosmetic method of treating skin, comprising adminstering topically the product obtainable by the process of claim
 32. 68. A compound according to formula 5, wherein A is a C₇-C₁₂ benzylic group.
 69. A compound according to formula 4, wherein A is a C₇-C₁₂ benzylic group and C is selected from monofunctional aliphatic hydroxyl protecting groups.
 70. A compound according to formula 3, wherein A is selected from a C₁-C₅ alkyl group or a C₇-C₁₂ benzylic group and C is selected from mono functional aliphatic hydroxyl protecting groups.
 71. A compound according to formula 2, wherein C is selected from monofunctional aliphatic hydroxyl protecting group.
 72. A compound according to formula 10, wherein A is a C₇-C₁₂ benzylic group, D is ethylene dioxy and X is halogen.
 73. A compound according to formula 11, wherein A is a C₇-C₁₂ benzylic group and D is ethylene dioxy. 